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SARS-Cov-2 Diagnosis: a Single-Centre Experience

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SARS-Cov-2 Diagnosis: a Single-Centre Experience JOURNAL of MEDICINE and LIFE JML | ORIGINAL ARTICLE SARS-CoV-2 diagnosis: a single-centre experience Ion Maruntelu 1, 2, Andreea Mirela Caragea 1, 2, Maria Tizu 1, 2, Ileana Constantinescu 1, 2 * Author Affiliations: * Corresponding Author: 1. Department of Immunology and Immunology of Transplant, Ileana Constantinescu, Professor, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania Centre of Immunogenetics and Virology, 2. Centre of Immunogenetics and Virology, Fundeni Clinical Institute, Fundeni Clinical Institute, 258 Fundeni Avenue, Bucharest, Romania Bucharest, 022328, Romania. Phone: +40744341984 E-mail: [email protected] DOI 10.25122/jml-2021-0064 Dates ABSTRACT Received: 18 February 2021 The coronavirus disease 2019 (COVID-19) was declared a pandemic Accepted: 27 March 2021 by the World Health Organization (WHO) on the 11th of March 2020. In Romania, there have been 983,217 confirmed cases and 24,386 deaths. We aim to show our experience at the Fundeni Clinical Institute in the diagnosis of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection in both patients and health care personnel. Swab samples were collected for extraction of the SARS-CoV-2 RNA from 29380 patients and health care personnel. We have combined three real-time reverse transcription-polymerase chain reaction (RT- PCR) assays for the qualitative detection of SARS-CoV-2. Also, the presence of IgG against SARS-CoV-2 nucleoprotein was analyzed in 1068 patients and clinical staff using the chemiluminescence method. Other 50 people were screened post-vaccination for the presence of SARS-CoV-2 antibodies against the spike (S) protein, using the chemi- luminescence method as well. The majority of confirmed cases were in adults, 71.3% of cases being registered in people aged 30-69 years. Most patients diagnosed with SARS-CoV-2 infection (83%) were ad- mitted to the gastroenterology, hematology, and surgery wards. Our study showed that one-third of people developed antibodies against the nucleocapsid of SARS-CoV-2. SARS-CoV-2 IgG seropreva- lence does not vary by gender or age. Also, we noticed the presence of antibodies against the SARS-CoV-2 spike protein in all 50 people post-vaccination that were tested two weeks after the second dose. Due to the increasing number of infected patients with SARS-CoV-2, the new coronavirus pandemic involves a sustained testing effort for an accurate virological diagnosis in both direct and indirect diagnosis. KEYWORDS: COVID-19, SARS-CoV-2, RT-PCR, anti nucleocapside, anti-protein S, patients and healthcare workers. ABBREVIATIONS: Ig – immunoglobulin; RNA – ribonucleic acid; RT- PCR – reverse transcription-polymerase chain reaction; WHO – World Health Organization. © 2021 JOURNAL of MEDICINE and LIFE. VOL: 14 ISSUE: 2 MARCH-APRIL 2021 257 JOURNAL of MEDICINE and LIFE INTRODUCTION In December 2019, many people with acute respiratory syndrome were identified in Wuhan, a city from Hubei, China [1]. In the following months, a similar acute respiratory disease has also been reported worldwide [2]. The World Health Organization (WHO) named the disease coronavirus disease 2019 (COVID-19) and declared it a pandemic in the next few months [3]. The International Committee on Taxonomy of Viruses renamed 2019-nCoV to Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). This virus was detected in different secretions from infected cases (i.e., throat swabs, conjunctival swabs, sputum) [4]. Thus, the main mode of transmission is from one person to another through different types of secretions. People of all ages could be infected, but the risk for complications increases with age [8] and immunological status. Around the world, 71.3 million people have been infected with SARS- CoV-2, and more than 1.6 million people have died from the disease, according to the WHO [5]. At the time of writing, there have been 983,217 confirmed cases of COVID-19 and 24,386 deaths in Romania [5]. Full-genome sequencing and phylogenetic analysis indicated that SARS-CoV-2 is a member of the coronavirus family. Determina- tion of SARS-CoV-2 partial genome sequence allowed the development of real-time reverse transcriptase-polymerase chain reaction (RT-PCR) methods for the detection of SARS-CoV-2 [6, 7]. RT-PCR tests were based on RNA-dependent RNA polymerase (RdRp), ORF1ab, E, N, and S genes of the SARS-CoV-2 genome [8–11]. We aimed to share our experience at the Fundeni Clinical Institute in the diagnosis of SARS-CoV-2 infection in both patients and health care personnel and to reveal the specific immunization. MATERIAL AND METHODS This study was carried out at the Fundeni Clinical Institute, Bucharest, Romania. Both nasal and oropharyngeal specimens were collect- ed from patients and health care workers, and electronic medical records provided various epidemiological and clinical data. Real-time reverse transcription-polymerase chain reaction assay for COVID-19 Swab samples were collected for the extraction of SARS-CoV-2 RNA from 23338 patients and clinical staff. We have approached direct virological diagnosis using three real-time RT-PCR assays for the qualitative detection of SARS-CoV-2 (Bosphore, Anatolia Geneworks, Turkey; Seegene, South Korea; GeneXpert, Cepheid, United States of America). These three assays were used to validate the methods (interassay validation) and also to cover the emergencies for the transplantation procedure. Bosphore Anatolia Geneworks products targeted the orf1 ab genome region, N gene (added in the last version of the kit) and E gene. Seegene products targeted the N gene, RNA-dependent RNA polymerase –RdRp and E gene, while GeneXpert SARS-CoV-2 tests detected the E and N2 genes in less than an hour. The GeneXpert SARS-CoV-2 test was used to screen patients before the transplantation procedure. The RT-PCR tests were settled according to the manuscript instructions. Also, the presence of IgG against SARS-CoV-2 nucleoprotein was analyzed in 1068 patients and clinical staff using the chemiluminescence method (SARS-CoV-2 IgG assay, Abbott, Illinois, United States of America). We have started to investigate the anti-spike antibody onset after vaccination with Pfizer, AstraZeneca, and Moderna vaccines. Statistical analysis Statistical analysis was done using the Statistical Package for the Social Sciences (SPSS) version 13.0 software (SPSS Inc). The categor- ical variables are summarized numbers and percentages. RESULTS We have included in our study all the patients that were tested for before their admission to the hospital. Also, 2382 individuals that are part of the medical staff of the Fundeni Clinical Institute were analyzed. All participants were tested for the SARS-CoV-2 infection. The median age of the patients was 55.72 years, and 52.21% of them were men. The median age was 46 years for the clinical staff, and 70.44% of them were women. Patients from the entire country usually come to be treated in our Institute, but most of the patients were from Bucharest-Ilfov (35.5%). The highest number of SARS-CoV-2 tests was requested from the gastroenterology, hematology, oncology, neurology, and urology wards, which represented 75.49% of all tests (Table 1). Regarding the patients, the positive rate of the RT-PCR tests was 7.23%. All the patients positive for the SARS-CoV-2 infection had one or more comorbidities (diabetes, obesity and others) and were aged between 1 and 101 years old. The biggest incidence of positive cases (72.42%) was seen in individuals aged 39–74 years. The highest number of positive cases was noticed in patients admitted to the hematology and oncology departments, and we have correlated this to their secondary immunodeficiencies (Table 1). © 2021 JOURNAL of MEDICINE and LIFE. VOL: 14 ISSUE: 2 MARCH-APRIL 2021 258 JOURNAL of MEDICINE and LIFE Table 1. Positive and negative RT-PCR tests in the most important wards of the Fundeni Clinical Institute. Row Labels Negative RT-PCR Positive RT-PCR Sum of grand total RT-PCR Gastroenterology 2345 73 2418 General Surgery 989 58 1047 Hem cell stem transplantation 102 16 118 Hematology 1896 161 2057 Intensive Care Unit 63 18 81 Internal Medicine 1267 72 1339 Kidney transplantation 86 6 92 Nephrology 844 46 890 Neurology 1177 77 1254 Oncology 1591 119 1710 Pediatric 343 18 361 Urology 2316 74 2390 Three hundred eighty-nine healthy individuals who were part of the medical staff were also positive for the SARS-CoV-2 infection. Most of the infected medical workers (212) came from the intensive care unit, general surgery, gastroenterology, and hematology depart- ments. The rate of positive RT-PCR results was higher in the case of female health care workers (n=293) than males (n=96). Also, 1068 patients and health care personnel were screened for the presence of SARS-CoV-2 IgG anti-nucleocapsid antibodies using the chemiluminescence method (Table 2). The test for SARS-CoV-2 IgG antibodies revealed that 253 patients and 157 health workers of the Institute developed IgG antibodies against the virus. Other 50 people (patients and medical staff) were screened post-vaccination for the presence of SARS-CoV-2 antibodies against the spike (S) protein. All of them developed antibodies against SARS-CoV-2 spike protein. DISCUSSION SARS-CoV-2 targets alveolar epithelial cells and enterocytes, leading to Covid-19 [12, 13]. The disease is predominantly characterized by respiratory symptoms and may progress to bilateral interstitial pneumonia, respiratory failure, or acute respiratory distress syndrome (ARDS) [14]. According to a WHO report from March 2021, there have been 983,217 confirmed cases of COVID-19 and 24,386 deaths in Romania [5]. At the Fundeni Clinical Institute, a significant number of RT-PCR tests for the SARS-CoV-2 infection were performed for both admit- ted patients and medical personnel. Our experience in the interpretation of RT-PCR results revealed at least four possible profiles.
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